Autolysins have been proposed to play important roles in cell morphogenesis, competence for genetic transformation, and antibiotic induced lysis. They have not, however, been thoroughly investigated at the molecular or genetic level. The long-term goal of our research is to elucidate the mechanism of regulation of autolysins in Staphylococcus aureus. We have isolated two autolytic-deficient mutants (lyt-1 and lyt- 2) of S. aureus by transposon mutagenesis. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic band compared to over 10 autolytic bands in the parent strain. On the basis of this data we believe that the mutation is either in a master regulatory gene, which controls the transcription of various lytic genes present in an operon or in multiple operons; or in a structural gene, which is responsible for the synthesis of the majority of autolytic bands, derived from a single polypeptide after cleavage and post-translational modifications. The objective of this study is to determine the mechanism of lyt-1 and lyt-2 alteration of lytic activity by: 1) cloning, sequencing and mapping of the mutated gene: This will be achieved by cloning the 5' flanking sequences of the mutated gene and probing a genomic library of S. aureus followed by deletion, complementation, nucleotide sequencing, and Northern analysis. A physical mapping technique will be used to locate the gene on the S. aureus chromosome. 2) Biochemical characterization: The gene will be overexpressed in E. coli to study the biochemical properties of the gene product. In vitro and in vivo analyses will be performed to characterize the gene product. 3) Regulation of the mutated gene in S. aureus: Since both the mutants have transposon (Tn917-lacZ) inserted in the proper orientation, the effect of various factors on the expression these mutants will be determined by measuring the activity of beta-galactosidase reporter gene. 4) Immunological and biochemical studies: Antibodies prepared against the purified product will be used to localize the gene product in the cell and to determine whether multiple lytic bands are immunologically related. The results of these studies should not only lead to the mechanism of autolytic gene(s) regulation but also improve our understanding of staphylococcus antibiotic resistance, which may have practical significance.